The proposed studies build on our previous findings indicating that interactive T- and B-like lymphocytes are basic features of the adaptive immune system in both jawless and jawed vertebrates, although jawless vertebrates (lampreys and hagfish) generate variable lymphocyte receptors (VLR) for antigen recognition by recombinatorial conversion of incomplete germline VLR genes (VLRA, VLRA and VLRC) into fully assembled VLR genes using diverse leucine-rich repeat (LRR) donor sequences. Recent studies indicate that while VLRA gene assembly coincides with expression of cytidine deaminase 1 (CDA1) within the lymphoepithelial thymoid gill region, coincident VLRB and CDA2 expression instead occurs primarily in hematopoietic tissues. A comprehensive analysis is proposed to elucidate the development, distribution and function of the B-like VLRB lymphocytes in lampreys. The overall goals are to better understand B cell biology in a jawless vertebrate, in particular the mechanism of generating VLRB antibody diversity, and to harness the lamprey antibody system for biomedical purposes. An efficient newly-developed scheme for lamprey immunization will be coupled with subsequent high-throughput screening of VLRB cDNA libraries from immunized animals to obtain specific monoclonal VLRB antibodies with high binding affinity for model antigens of biomedical significance. The first specific aim is to elucidate the sequence diversity and possibility of VLRB antibody affinity maturation during lamprey B cell responses to primary and booster immunizations with model protein antigens. The second specific aim is to determine the lamprey CDA2 structure, functional potential, and expression as a function of the differentiation status and tissue localization of VLRB lineage cells. The third specific aim is to produce recombinant VLRB antibodies with novel specificities for use as diagnostic reagents for medically important infectious agents and tumor cell types. These interrelated studies will provide insight into the evolution of the alternative B cell types f jawless and jawed vertebrates and will also yield novel monoclonal lamprey antibodies with specificity for infectious agents and tumor cell antigens of humans for use as diagnostic and disease-monitoring reagents.